L-arabinitol 4-dehydrogenases (LAD) from Neurospora crassa and their uses in production of sugar alcohols including xylitol from arabinose containing media are disclosed.
Lignocellulosic biomass represents a renewable resource that is available in sufficient quantities from the corn wet-milling industry to serve as a low-cost feedstock. Some sources, particularly corn fiber, contain significant amounts of L-arabinose, an abundant pentose sugar second only to D-xylose in biomass composition. However, utilization of the L-arabinose content from hemicellulose hydrolysates for production of valued products has resulted in limited success. The inability of many yeasts and fungi to ferment L-arabinose appears to be a consequence of inefficient or incomplete assimilation pathways for this pentose sugar. It has also been suggested that the cofactor imbalance necessary for the catabolism of L-arabinose also plays a factor. Recently some progress has been made with the overexpression of either the bacterial utilization pathway or the fungal pathway for production of ethanol from L-arabinose. One benefit of utilizing the fungal pathway is that the intermediate xylitol is also formed, which is a five-carbon sugar alcohol that has attracted much attention because of its potential as a natural food sweetener, a dental caries reducer, and a sugar substitute for diabetics.
Xylitol is a pentitol and is used not only as a sweetener but also as a platform chemical for the production of industrially important chemicals. Studies have shown that among sugar substitutes, xylitol is one of the most promising candidates for application in a wide range of products due to several favorable properties. These include anti-cariogenicity, suitability for use by diabetic patients, and good gastrointestinal tolerance, in addition to possibly preventing osteoporosis and ear infections. In spite of its advantages, the use of xylitol is currently limited and falls well short of another, cheaper sugar alternative, sorbitol in the billion dollar polyol market. Other than its use as a sweetener, xylitol is also an industrially important chemical, and the US Department of Energy (DOE) has named it among one of their top 12 platform chemicals from agricultural sources.
L-arabinitol 4-dehydrogenase (LAD, EC 1.1.1.12), a common enzyme found in yeasts and filamentous fungi, catalyzes the second step of the recently elucidated fungal L-arabinose metabolic pathway by oxidizing L-arabinitol to L-xylulose with concomitant NAD+ reduction. LAD is purportedly a fungal orthologue of the eukaryotic sorbitol dehydrogenase (SDH) and belongs to the family of zinc-containing alcohol dehydrogenases. Several LADs have successfully been cloned and expressed. However, they are not optimal for in vitro enzymatic production of xylitol due to their poor stability and/or activity.
L-arabinose is a major constituent of some plant materials, up to 15% of materials such as wheat bran and corn cob hulls, so that L-arabinose processing is of relevance for micro-organisms using plant material as a carbon source. The bacterial pathway for L-arabinose catabolism is known. It includes an isomerase, a kinase, and an epimerase that sequentially convert L-arabinose to L-ribulose, L-ribulose 5-phosphate, and D-xylulose 5-phosphate. D-xylulose 5-phosphate is an intermediate of the pentose phosphate pathway. There is also a pathway for fungi that may include five enzymes, aldose reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, xylitol dehydrogenase, and xylulokinase. The intermediates are, in this order: L-arabinitol, L-xylulose, xylitol and D-xylulose.